Immortalized avian cell lines and use thereof

ABSTRACT

The present invention relates to specific immortalized avian cell lines expressing telomerase reverse transcriptase (TERT), and exhibiting distinct biologics production patterns. More particularly, the present invention relates to immortalized avian cell line capable of either amplifying Flaviviridae but not capable of amplifying Vaccinia virus strain Copenhagen (VV-COP) nor Modified Vaccinia virus Ankara (MVA), or capable of amplifying both Flaviviridae and Poxviridae. The invention further relates to the use of said immortalized avian cell lines and related methods for producing biologics, including viruses and proteins.

TECHNICAL FIELD

The present invention pertains to the field of cell lines for theproduction of biologics, including viruses and proteins. In particular,the invention relates to specific immortalized avian cell linesexpressing telomerase reverse transcriptase (TERT), and exhibitingdistinct biologics production patterns. More particularly, the presentinvention relates to immortalized avian cell line capable of eitheramplifying Flaviviridae but not capable of amplifying Vaccinia virusstrain Copenhagen (VV-COP) nor Modified Vaccinia virus Ankara (MVA), orcapable of amplifying both Flaviviridae and Poxviridae. The inventionfurther relates to the use of said immortalized avian cell lines andrelated methods for producing biologics, including viruses and proteins.

BACKGROUND OF THE INVENTION

Avian cells have been used for years for the production of viralvaccines. For instance, embryonated chick eggs are able to support thereplication of a wide range of human and animal viruses includingnotably attenuated viruses which have impaired potential to replicate inhuman or mammalian cells. Embryonated chick eggs used for human viralvaccine production must be certified to be free of a defined set ofviral and bacterial contamination. (specific pathogen-free or SPF).Embryonated chick eggs are expensive and can constitute up to 40% of thecost of viral vaccines. A vaccine lot cannot be released until the SPFsupplier verifies that the parental chickens for the embryonated chickeggs used to manufacture the vaccine lot were completely free of anydisease. This uncertainty adds a significant cost to the preparation ofthese vaccines. In addition, the large-scale processes for infectingeggs and maintaining virus growth are time consuming and sometimesinconsistent across different vaccine batches.

With the development of cell culture techniques vaccine manufacturershave replaced embryonated chick eggs with isolated chicken embryofibroblasts (CEFs). Most Flaviviridae vaccines are for instancemanufactured from CEFs. The Flaviviridae Yellow fever viral vaccinesrecommended by the World Health Organization (WHO) are from 17D strainand substrains, which have minor differences in nucleotide sequences andequivalent immunogenicity (CAMACHO L.A.B. et al., Rev Saude Publica38(5):671-8 (2004); DOS SANTOS C. N., Virus Res. 35:35-41 (1995)).Experiments regarding Flaviviridae viral production process haveresulted in the establishment of an efficient methodology for theproduction of a Yellow fever viral vaccine in CEFs using the 17DD virusstrain (FREIRE M. S., Vaccine 23, 2501-2512 (2005)). While the use ofCEFs improves the safety profile, efficiency and reliability of themanufacturing process, it also highly time consuming and furtherincreases costs. The production of CEFs depends indeed on theavailability of SPF eggs. The CEFs are prepared from SPF eggs by mincingembryos to establish and amplify viable cells. Typical for primaryanimal cells the fibroblasts suffer senescence: the doubling timeincreases with passaging and eventually all cells die. This processoccurs after about 20 passages. This limited live span constraints toperform a complete set of safety tests for each lot of CEFs.

Due to the limitations of embryonated chicken eggs and CEFs aspreviously described for manufacturing viral vaccines, the search fornew cell culture systems have drawn considerable attention over the pastdecades. In this respect immortalized cell lines are highly attractive.Immortalized cell lines can be maintained or frozen from batch to batchon the production site and are always available for a new productionprocess. Moreover as they are confined at the production plant, they areless subject to contamination by exogenous contaminant. Their use allowsa drastic reduction of the manual manipulation needed for the productionprocess. All these properties lead to a reduction of the price and ofthe duration of the production process as well as a diminution of thepotential contamination. Immortalized cell lines can moreover be fullycharacterized and are thus totally compliant with the good laboratorypractice and the requirements of the different medical agencies. As anexample, VERO cell line has been described as alternative candidate forFlaviviridae vaccine production in replacement of the approvedembryonated chicken eggs. The live attenuated Japanese encephalitisvirus vaccine ChimeriVax™-JEV was propagated in VERO cells cultured inmedia supplemented with fetal bovine serum (MONATH et al., Biologicals,33:131-144 (2005)). Similarly, MATEU et al. (Trans. R. Soc. Trap. Med.Hyg., 101(3):289-98 (2007)) and TORINIWA et al. (Vaccine,4;26(29-30):3680-9 (2008)) have respectively produced Yellow fever virusvaccine and Japanese encephalitis virus vaccine in VERO cell line. Theapplication of immortalized cell lines is moreover not limited to theviral vaccines they are designed for but may be extended to recombinantproteins. For example, both antibodies and influenza viral vaccines canbe propagated on PER.C6® cell line (ECACC number 96022940), a humanfetal retinoblast cell line immortalized by transfection with an E1minigene of adeno virus type 5. (FALLAUX F. J. et al., Hum. Gene Ther. 9:1909-17 (1998); PAU M. G. et al., Vaccine 19, 2716-2721 (2001); YALLOPC. et al., Animal Cell Technology meets Genomics, 533-536 (2005)).

However alternative immortalized cell lines are still needed.WO2007/077256 provides methods for immortalizing primary avian cells bytransfection with the Cairina moschata telomerase reverse transcriptase(TERT) nucleic acid molecule either by targeted or random insertion ofthe TERT nucleic acid molecule into the Cairina moschata primary aviancells genome. Based on said methods, especially by random insertion, wehave now identified new immortalized avian cell lines which havedistinguished properties from one another.

DISCLOSURE OF THE INVENTION

As used throughout the entire application, “a” and “an” are used in thesense that they mean “at least one”, “at least a first”, “one or more”or “a plurality” of the referenced components or steps, unless thecontext clearly dictates otherwise.

As used throughout the entire application, “and/or” wherever used hereinincludes the meaning of “and”, “or” and “all or any other combination ofthe elements connected by said term”.

As used throughout the entire application, “comprising” and “comprise”are intended to mean that the products, compositions and methods includethe referenced components or steps, but not excluding others.“Consisting essentially of when used to define products, compositionsand methods, shall mean excluding other components or steps of anyessential significance. Thus, a composition consisting essentially ofthe recited components would not exclude trace contaminants andpharmaceutically acceptable carriers. “Consisting of” shall meanexcluding more than trace elements of other components or steps.

As used throughout the entire application, “about” or “approximately” asused herein means within 20%, preferably within 10%, and more preferablywithin 5% of a given value or range.

As used throughout the entire application, an “immortalized avian cellline” refers to an avian cell line that proliferates in culture beyondthe Hayflick limit (HAYFLICK L., Clin. Geriatr. Med. 1(1):15-27 (1985)).More particularly, an “immortalized avian cell line” refers to an aviancell line that is capable of growing in culture for greater than 30passages that maintain a doubling time in culture of about 1 to about 2days and have been in continuous culture for greater than about 6months. An avian cell line is considered immortalized after about 20 toabout 25 passages in culture. Immortalized avian cells aredifferentiated from transformed cells in that unlike transformed cells,immortalized avian cells are growth arrested (i.e. avian cells areconfluent and subject to contact inhibition) and have a homogenousfibroblast like morphology.

As used throughout the entire application, the terms an “immortalizedavian cell line able of amplifying a virus” mean that an immortalizedavian cell line of the invention is able after infection by a virus toincrease the amount of said virus due to a productive viral replicationof the virus in the infected cells. The term “reproductive replication”refers to the fact that the said virus replicates in the immortalizedavian cell line to such an extent that infectious progeny virus isproduced, wherein the ratio of output virus to input virus is above 1.In other words, “able of amplifying a virus” means that the ratio ofoutput virus to input virus should be above 1.

As used throughout in the entire application, “Flaviviridae” includeFlaviviruses, Pestiviruses, Hepaciviruses, GB virus A, GB virus A-likeagents, GB virus-B and GB virus-C (also called hepatitis G virus).Classification, genome organisation and replication cycle ofFlaviviridae has been well described (LINDENBACH B. D. at al., in D. M.Knipe and P. M. Howley, Fields Virology 5^(th) Edition, Eds.Lippincott-Raven Publishers, Philadelphia (2007)).

As used throughout in the entire application, “Flaviviruses” include the“mosquito-borne virus cluster”, the “tick-borne virus cluster” and the“no-vector cluster” (KUNO G. et al., J. Virol. 72, 73-83 (1998)).

The “mosquito-borne virus cluster” includes Dengue virus, Japaneseencephalitis virus, Murray Valley encephalitis virus, St. Louisencephalitis virus, West Nile virus, Yellow fever virus, Kunjin virus,Rocio virus and Ilheus virus.

Dengue virus (DENV) strains are divided into the four serotypes DEN-1,DEN-2, DEN-3 and DEN-4. Information on the nucleotide sequences of thegenomes of DENVs can also be obtained from publicly accessible genedatabases such as GenBank: DEN-1 virus (e.g. GenBank accession numberM23027); DEN-2 virus (e.g. GenBank accession number M19197; NC-001474);DEN-3 virus (e.g. GenBank accession number M93130); DEN-4 virus (e.g.GenBank accession number M14931).

Japanese encephalitis virus (JEV) includes the strains: P3, SA14, S892,GP78, ThCMAr4492, ThCMAr6793, JaGAr01, Jaoars982, Subin, KE-093/83,Nakayama wild-strain, Nakayama-RFVL, Nakayama-Yoken, LNDG07-02,LNDG07-16 and K94P05. Information on the nucleotide sequences of thegenomes of JEVs can also be obtained from publicly accessible genedatabases such as GenBank: e.g. GenBank accession number AF045551.

West Nile virus (WNV) strains are including genotypes NY99 and WN02.Information on the nucleotide sequences of the genomes of WNVs can alsobe obtained from publicly accessible gene databases such as GenBank:e.g. GenBank accession number M12294; NC-001563.

The Yellow fever virus (YFV) strains include the strains: Asibi, Frenchviscerotropic virus (FVV), B4.1, Rendu, Dak1279, 17D, 17DD, 17D-204,Colombia 88, F-204 and C-204 (HAHN et al., Proceedings of the NationalAcademy of Sciences USA 84, 2019-2023 (1987); WANG et al., J. of Gen.Virol. 76, 2749-2755 (1995); BALLINGER-CRABTREE & MILLER, J. of Gen.Virol. 71, 2115-2121 (1990)); WANG H. et al., J. of Gen. Virol. 78,1349-1352 (1997); CAMACHO L.A.B. et al., Rev Saude Publica 38(5):671-8(2004); DOS SANTOS C. N., Virus Res. 35:35-41 (1995); GALLER R. et al.,Braz. J. Med. Biol. Res. volume 30(2), 157-168 (1997). The Yellow fevervirus (YFV) has been studied at the genetic level (RICE et al., Science229:726-733 (1985)) and information correlating genotype and phenotypehas been established (MARCHEVSKY et al., Am. J. Trop. Med. Hyg. 52:75-80(1995)). Information on the nucleotide sequences of the genomes of YFVscan also be obtained from publicly accessible gene databases such asGenBank: e.g. GenBank accession number X03700; NC-002031.

The “tick-borne virus cluster” refers to Tick-borne encephalitis virus(TBEV) (also called Tick-borne meningoencephalitis virus) which includesthree subtypes: the Western subtype (also called Central Europeanencephalitis virus), the Far Eastern subtype (also called Russianspring/summer encephalitis virus) and the Siberian subtype (KAISER andREINHARD, Infectious Disease Clinics of North America. 22(3):561-575(2008)). Information on the nucleotide sequences of the genomes of TBEVscan also be obtained from publicly accessible gene databases such asGenBank: e.g. GenBank accession number U27495, NC-001672. TBEV alsoincludes the viable chimeric vaccine was constructed which contained theC-preM-E or preM-E structural protein genes of a virulent Far EasternRussian TBEV with the remaining nonstructural protein genes and 5′- and3′-noncoding sequences derived from DEN4 [TBEV(CME)/DEN4 andTBEV(ME)/DEN4, respectively] (PLETNEV et al., Proc. Natl. Acad. Sci.U.S.A. 89:10532-10536, (1992)).

The “no-vector cluster” includes Apoi virus, cell fusing agent virus,San Perlita virus, Jutiapa virus, Montana myotis leukoencephalitisvirus, Modoc virus, Cowbone Ridge virus, Sal Vieja virus, Bukalasa batvirus, Dakar bat virus, Rio Bravo virus, Carey Island virus, Phnom Penhbat virus and Batu Cave virus (KUNO G. et al., J. Virol., 72 73-83(1998)). Information on the nucleotide sequences of the genomes ofno-vector viruses can also be obtained from publicly accessible genedatabases such as GenBank. For instance, for Apoi virus: e.g. GenBankaccession number AF160193, NC-003676.

As used throughout in the entire application, “Pestiviruses” includeBorder disease virus (BDV), Bovine viral diarrhea virus (BVDV) andclassical swine fever virus (CSFV). BDV includes the 3 genotypes BDV-1to -3 (BECKER et al. Virology 311 96-104 (2003)). BVDV includes BVDVtype 1 (BVDV-1) and BVDV type 2 (BVDV-2) (HEINZ et al. in Seventh Reportof the International Committee on Taxonomy of Viruses, 859-868, Editedby M. H. V. Van Regenmortel, C. M. Fauquet, D. H. L. Bishop & 8 othereditors. San Diego: Academic Press (2000)).

As used throughout in the entire application, “Hepaciviruses” includehepatitis C virus (HCV). Extensive phylogenetic analyses have led to theclassification of HCV isolates into 6 major genotypes (1 to 6)containing different subtypes (a, b, c, etc. . . . ) (SIMMONS et al.,Hepatology 42, 962-973 (2005)). Exemplary HCV isolates of genotype 1ainclude without limitation, HCV-1 (CHOO et al., Proc. Natl. Acad. Sci.USA 88, 2451-2455 (1991)), -J1 (OKAMOTO et al., Nucleic Acids Res. 20,6410-6410 (1992)) and -H (INCHAUSPE et al., Proc. Natl. Acad. Sci. 88,10292-10296 (1991)). Exemplary HCV isolates of genotype 1b includewithout limitation, HCV-JA (KATO et al., Proc. Natl. Acad., Sci. 87,9524-9528 (1990)) and BK (TAKAMIZAWA et al., J. Virol. 65, 1105-1113(1991)). Exemplary HCV isolates of genotype 1c include withoutlimitation, HCV-G9 (OKAMOTO et al., J. Gen. Viral. 45, 629-635 (1994)).Exemplary HCV isolates of genotype 2a include without limitation, HCV-J6(OKAMOTO et al., J. Gen. Virol. 72, 2697-2704 (1991)). Exemplary HCVisolates of genotype 2b include without limitation, HCV-J8 (OKAMOTO etal., Virology 188, 331-341 (1992)). Exemplary HCV isolates of genotype2c include without limitation, HCV-BEBE1 (NAKO et al., J. Gen. Virol.141, 701-704 (1996)). Exemplary HCV isolates of genotype 3a includewithout limitation, HCV-NZL1 (SAKAMOTO et al., J. Gen. Virol. 75,1761-1768 (1994)). Exemplary HCV isolates of genotype 3b include withoutlimitation, HCV-Tr (CHAYAMA et al., J. Gen. Virol. 75, 3623-3628(1994)). Exemplary HCV isolates of genotype 4a include withoutlimitation, HCV-ED43 (CHAMBERLAIN et al., J. Gen. Viral. 78, 1341-1347(1997)). Exemplary HCV isolates of genotype 5a include withoutlimitation, HCV-EUH1480 (CHAMBERLAIN et al., Biochem. Biophys. Res.Commun. 236, 44-49 (1997)). Exemplary HCV isolates of genotype 6ainclude without limitation, HCV-EUHK2 (ADAMS et al., Biochem. Biophys.Res. Commun. 234, 393-396 (1997)).

As used throughout in the entire application, “Poxviridae” includeOrthopoxviruses, Capripoxviruses, Avipoxviruses, Parapoxviruses andLeporipoxviruses, and derivatives thereof.

Orthopoxviruses include Buffalopoxvirus, Camelpoxvirus, Cowpoxvirus,Ectromelia virus, Monkeypoxvirus, Rabbitpoxvirus, Variola virus,Vaccinia virus (VV) and its derivatives such as for instance ModifiedVaccinia virus Ankara (MVA).

Capripoxviruses include sheeppox virus, goatpox virus and lumpy skindisease virus.

Avipoxviruses include Canarypoxvirus and Fowlpoxvirus.

Parapoxviruses include pseudocowpox, arapoxvirus ovis and orf virus.

Leporipoxviruses include Myxoma virus.

Sequences of the genome of various Poxviridae are available in the art,for example, the VV Western reserve, VV Copenhagen, Modified Vacciniavirus Ankara, Cowpoxvirus, Canarypoxvirus, Ectromelia virus, Myxomavirus genomes are available in Genbank (accession number NC_(—)006998,M35027, U94848, NC_(—)003663, NC_(—)005309, NC_(—)004105, NC_(—)001132respectively).

As used throughout in the entire application, “Vaccinia virus” (VV)includes the VV strains: Dairen I, IHD-J, L-IPV, LC16M8, LC16MO, Lister,LIVP, Tashkent, WR 65-16, Wyeth, Ankara, Copenhagen (COP) (GOEBEL et al.(1990); Genbank accession number M35027.1), Tian Tan, Western Reserve(WR), Modified Vaccinia virus Ankara (MVA), VV comprising a defectiveJ2R gene (see WEIR and MOSS 1983; Genbank accession number AAA48082), VVcomprising a defective F2L gene (see WO2009/065547), W comprisingdefective I4L and/or F4L gene(s) (see WO2009/065546), and derivativesthereof.

As used throughout in the entire application, “Modified Vaccinia virusAnkara (MVA)” refers to the highly attenuated VV generated by serialpassages on CEFs of the VV strain Ankara (MAYR A. et al., Infection 3,6-14 (1975)) and derivatives thereof. The MVA virus was deposited beforeCollection Nationale de Cultures de Microorganismes (CNCM) underdepositary N° I-721. The MVA is fully described in SUTTER et MOSS (Proc.Natl. Acad. Sci. USA 89, 10847-10851 (1992)). The genome of the MVA hasbeen mapped and sequenced (ANTOINE et al., Virol. 244, 365-396 (1998)and is available in Genbank under accession number U94848).

According to a first embodiment, the present invention concerns a methodfor producing virus, comprising the steps of:

-   -   (a) providing an immortalized avian cell line;    -   (b) infecting said immortalized avian cell line with a virus to        be produced; and    -   (c) cultivating the said infected avian cell line under        conditions which are enabling virus amplification;        wherein said immortalized avian cell line is selected in the        group consisting of immortalized avian cell line deposited at        the European Collection of Cell Cultures (ECACC) under accession        number 08060502, an immortalized avian cell line deposited at        the ECACC under accession number 08060501 and derivatives        thereof.

According to another embodiment, the present invention relates to animmortalized avian cell line expressing avian telomerase reversetranscriptase (TERT) selected in the group consisting of an immortalizedavian cell line deposited at the European Collection of Cell Cultures(ECACC) under accession number 08060502, an immortalized avian cell linedeposited at the ECACC under accession number 08060501 and derivativesthereof. These cell lines have been generated from primary Cairinamoschata cells as disclosed in WO2007/077256 by random insertion of theavian TERT nucleic acid molecule (SEQ ID NO: 1) into the Cairinamoschata primary avian cells genome.

Surprisingly, these immortalized avian cell lines expressing aviantelomerase reverse transcriptase (TERT) have distinct properties, andmore particularly distinguish one from another by their virus strainamplification feature.

According to one special embodiment, the immortalized avian cell line ofthe invention is able of amplifying at least one Flaviviridae strainwhile being unable of amplifying Vaccinia virus strain Copenhagen(VV-COP) (GOEBEL et al. 1990; Genbank accession number M35027.1) norModified Vaccinia virus Ankara (MVA) (Collection Nationale de Culturesde Microorganismes (CNCM) under depositary N° I-721).

Detailed informations regarding assays used to determine whether animmortalized avian cell line is or not able of amplifying one specificvirus strain are given further below.

According to preferred embodiment, the immortalized avian cell line ofthe invention able of amplifying at least one Flaviviridae strain whilebeing unable of amplifying Vaccinia virus strain Copenhagen (W-COP) norModified Vaccinia virus Ankara (MVA) is an immortalized avian cell lineexpressing avian telomerase reverse transcriptase (TERT) deposited atthe European Collection of Cell Cultures (ECACC) under accession number08060502 or derivatives thereof.

According to preferred embodiment, the immortalized avian cell linedeposited at the European Collection of Cell Cultures (ECACC) underaccession number 08060502 or derivatives thereof is able of amplifyingat least one Flaviviridae strain selected in the group consisting ofYellow fever virus strain (YFV) and Japanese encephalitis virus strain(JEV), and more particularly in the group consisting of YFV 17D strain(e.g. ref. 507 at HPA Culture Collections) and JEV Nakayama wild strain(e.g. ref. 502 at HPA Culture Collections).

According to preferred embodiment, the immortalized avian cell linedeposited at the European Collection of Cell Cultures (ECACC) underaccession number 08060502 or derivatives thereof is able of amplifyingYellow fever virus strain (YFV) and Japanese encephalitis virus strain(JEV).

According to another special embodiment, the immortalized avian cellline of the invention is able of amplifying (i) at least oneFlaviviridae strain and (ii) at least one Poxviridae strain.

According to preferred embodiment, the immortalized avian cell line ofthe invention able of amplifying at least one Flaviviridae strain and atleast one Poxviridae strain is an immortalized avian cell lineexpressing avian telomerase reverse transcriptase (TERT) deposited atthe European Collection of Cell Cultures (ECACC) under accession number08060501 or derivatives thereof.

According to preferred embodiment, the immortalized avian cell linedeposited at the European Collection of Cell Cultures (ECACC) underaccession number 08060501 or derivatives thereof is able of amplifying(i) at least one Flaviviridae strain selected in the group consisting ofYellow fever virus strain (YFV) and Japanese encephalitis virus strain(JEV), and more particularly in the group consisting of YFV 17D strain(e.g. ref. 507 at HPA Culture Collections) and JEV Nakayama wild strain(e.g. ref. 502 at HPA Culture Collections), and (ii) at least onePoxviridae strain selected in the group consisting of Vaccinia virusstrain Copenhagen (VV-COP) (GOEBEL et al. 1990; Genbank accession numberM35027.1) and Modified Vaccinia virus Ankara (MVA) (Collection Nationalede Cultures de Microorganismes (CNCM) under depositary N° I-721).

According to preferred embodiment, the immortalized avian cell linedeposited at the European Collection of Cell Cultures (ECACC) underaccession number 08060501 or derivatives thereof is able of amplifyingYellow fever virus strain (YFV), Japanese encephalitis virus strain(JEV), Vaccinia virus strain Copenhagen (VV-COP) and Modified Vacciniavirus Ankara (MVA)

Derivatives of the deposited Cairina moschata immortalized avian celllines of the invention refer to Cairina moschata immortalized avian celllines derived from the deposited ones by, for example:

-   -   subcloning of the said deposited Cairina moschata immortalized        avian cell line (ECACC 08060502 or ECACC 08060501);    -   adaptation of the said deposited Cairina moschata immortalized        avian cell line (ECACC 08060502 or ECACC 08060501) to a specific        culture medium (e.g. medium allowing the growth of the cell line        in suspension) and/or to specific culture conditions (e.g.        temperature; % CO₂):    -   deletion or mutation of one or more genes of Cairina moschata        involved in saccharide fucose modification (HARUE IMAI-NISHIYA        et al., BMC Biotechnology (2007)) in order to produced        non-fucosylated proteins, and more particularly non-fucosylated        antibodies (according to preferred embodiment, said derivatives        of the deposited Cairina moschata immortalized avian cell line        are obtained by deletion or mutation of α1,6-fucosyltransferase        (FUT8) and/or GDP-mannose 4,6-dehydratase (GMD) genes;    -   deletion or mutation of one or more genes of Cairina moschata        involved in interferon resistance in order to reduce the immune        response of the cell line (according to preferred embodiment,        said derivatives of the deposited Cairina moschata immortalized        avian cell line are obtained by deletion or mutation of gene(s)        selected in the group consisting of STAT1 gene, STAT2 gene,        STAT3 gene and STAT5 gene);    -   overexpression of one or more Cairina moschata's anti-apoptotic        gene(s), or transformation with one or more exogenous        anti-apoptotic gene(s) in order to render the cell line more        resistant to the culture conditions, in particular for        maintaining confluence (according to preferred embodiment, said        anti-apoptotic gene(s) is selected in the group consisting of        p19E1B human adenovirus gene, bcl-2 gene, mcl-1 gene, Bcl-xL        gene, Bcl-w gene, a1 gene, ICP34.5 herpes simplex virus gene and        p35 baculovirus gene);    -   overexpression of one or more Cairina moschata's genes involved        in controlling the cell cycle using vectors which are suitable        for increasing the rate of proliferation (according to preferred        embodiment, said gene(s) involved in controlling the cell cycle,        is selected in the group consisting of p53 gene, p21 gene, p27        gene and p57 gene);    -   modifying the viral sensitivity spectrum of the cell line by        transformation with one or more genes which encode receptors for        the viruses of interest, with a view to multiplying these        viruses (according to preferred embodiment, said gene(s) which        encode receptors for the viruses of interest, is gene(s) which        encode measles virus CD46 receptor).

According to preferred embodiment said ECACC 08060502 derivatives areable of amplifying at least one Flaviviridae strain while being unableof amplifying Vaccinia virus strain Copenhagen (VV-COP) nor ModifiedVaccinia virus Ankara (MVA).

According to preferred embodiment said ECACC 08060501 derivatives arecapable of amplifying (i) at least one Flaviviridae strain and (ii) atleast one Poxviridae strain.

According to preferred embodiment, the immortalized avian cell lines ofthe invention are adherent cell lines.

According to another preferred embodiment, the immortalized avian celllines of the invention are non-adherent cell lines which proliferate insuspension, in presence or not of (micro)carriers. The (micro)carriersused according to the invention can be made of dextran, collagen,polystyrene, polyacrylamide, gelatine, glass, cellulose, polyethyleneand/or plastic. (Micro)carriers are commercially available such as e.g.Cytodex™ microcarriers (Pharmacia), Cytopore™ microcarriers (GEHealthcare Life Sciences), Hillex™ microcarriers (SoloHill Enginnering),Nunc 2D MicroHex™ microcarriers Nunclon™ (Thermo Fisher Scientific),ProNectin™ microcarriers (SoloHill Enginnering), Fibra-Cell™ discs (NewBrunswick Scientific), BioNocll™ microcarriers (Cesco Bioengineering)and CultiSpher-S™ microcarriers (Percell Biolytica).

The present invention also relates to the use of an immortalized aviancell line of the invention for the production of viruses and proteins.

As used throughout the entire application, “viruses” includes virusesselected in the group consisting of Flaviviridae, Poxviridae, fluviruses, Paramyxoviridae, adenovirus, adeno-associated virus (AAV),retrovirus (such as e.g. Rous sarcoma virus (RSV); humanimmunodeficiency virus (HIV)), hepadnaviruses (such as e.g. hepatitis Bvirus), herpes viruses (such as e.g. HSV-1; HSV-2), reoviruses (such ase.g. rotaviruses), coronaviruses (such as e.g. human SARS-CoV;HCoV-NL63), and alphaviruses (such as e.g. chikungunya; Ross river virus(RRV). The viruses can be wild type, attenuated, recombinant and/ortemperature sensitive viruses.

The Flaviviridae and Poxviridae have been defined above.

According to the invention, the Flaviviridae is a wild type, attenuated,recombinant and/or temperature sensitive Flaviviridae.

According to the invention, the Poxviridae is a wild type, attenuated,recombinant and/or temperature sensitive Poxviridae.

As used throughout the entire application, “attenuated virus” refers toany virus that has been modified so that its pathogenicity in theintended subject is substantially reduced. Preferably, the virus isattenuated to the point it is nonpathogenic from a clinical standpoint,i.e. that subjects exposed to the virus do not exhibit a statisticallysignificant increased level of pathology relative to control subjects.Several experimental approaches to attenuation of wild type Flaviviridaepathogens have been described (see e.g. PUGACHEV et al., Int J.Parasitol. 33:567-582 (2003)). For example, it has been found thatmutations in certain amino acids of the envelope proteins of chimericFlaviviridae including capsid and non-structural proteins of YFV andmembrane and envelope proteins of JEV, a DENV, or West Nile virusdecrease viscerotropism (see e.g. WO2003/103571 and WO2004/045529).

As used throughout the entire application, “recombinant virus” refers toa virus comprising an exogenous sequence inserted in its genome. As usedherein, “exogenous sequence” refers to a nucleic acid molecule which isnot naturally present in the parent virus. “Recombinant virus” can referto a virus consisting of a virus in which one or more structuralproteins has been replaced with foreign nucleic acid sequence ofeukaryotic, prokaryotic, viral origin (e.g. structural protein(s) of asecond virus). Therefore, according to the invention, “recombinantvirus” can be also indifferently called “chimeric virus” or “hybridvirus”. Notably, in the case of Flaviviridae, chimeric Flaviviridae canconsist of a first Flaviviridae (e.g. a YFV such as for instance YFV 17Dstrain as previously described) in which the prM and E proteins havebeen replaced with the prM and E proteins of a second virus (e.g. a JEV,WNV, DENV, St. Louis encephalitis virus or TBEV as previouslydescribed). ChimeriVax™ technology has been used to create chimericvaccine candidates against medically important Flaviviridae. It employsthe YFV 17D vaccine virus as a vector in which the prM-E genes arereplaced with the prM-E genes from a heterologous Flaviviridae such asJEV, DENV, WNV or St. Louis encephalitis viruses (MONATH et al., Vaccine20:1004-1018 (2002); PUGACHEV et al., Int. J. Parasitol. 33:567-582(2003); GUIRAKHOO et al., J. Virol. 78:4761-4775 (2004)). TheChimeriVax™-JEV vaccine comprising the prM-E genes from the SA14-14-2virus (i.e. live attenuated JEV vaccine used in China), was successfullytested in preclinical and Phase I and II clinical trials. Similarly,successful Phase I clinical trials have been conducted with aChimeriVax™-WNV vaccine candidate which comprises the prM-E sequencefrom a WNV (i.e. NY99 strain) with three specific amino acid changesincorporated into the E protein to increase attenuation (ARROYO et al.,J. Virol. 78:12497-12507 (2004)). As other examples, chimericFlaviviridae can also be chimeric Flaviviridae as described inWO2006/068307, WO2002/102828, EP0977587, WO98/37911, WO93/06214, U.S.Pat. No. 7,569,383 and WO01/39802.

Advantageously, the recombinant virus can further comprise the elementsnecessary for the expression of the exogenous sequence(s). The elementsnecessary for the expression comprise of the set of elements allowingthe transcription of a nucleotide sequence to RNA and the translation ofa mRNA to a polypeptide, in particular the promoter sequences and/orregulatory sequences which are effective in the cell to be infected bythe recombinant virus, and optionally the sequences required to allowthe excretion or the expression at the surface of the cells for saidpolypeptide. These elements may be inducible or constitutive. Of course,the promoter is adapted to the recombinant virus selected and to thehost cell. The literature provides a large amount of informationrelating to such promoter sequences. The elements necessary can, inaddition, include additional elements which improve the expression ofthe exogenous sequence or its maintenance in the host cell. There may bementioned in particular the intron sequences, secretion signalsequences, nuclear localization sequences, internal sites forreinitiation of translation of the IRES type, poly A sequences fortermination of transcription.

As used throughout the entire application, “temperature sensitive virus”refers to a virus derivative which has an impaired growth at or above acertain temperature at which the wild type has a normal growth. Asexamples, the temperature sensitive viruses as described in BOYD O. etal. (Virology Apr. 10; 399(2):221-30 (2010)), EP 0 157 528 (smallpoxtemperature sensitive virus), and DRILLIEN R. et al. (Virology 119,372-381 (1982)), can be cited.

According to the invention, “Flu viruses” (also called influenzaviruses) includes influenza type A virus and its subtypes, influenzatype B virus and influenza type C virus. “Subtypes of influenza type Avirus” include the different combinations of HA and NA proteinspossible. 19 classes of NA proteins (classified H1-H15) and 9 classes ofNA proteins (classified N1-N9) have been identified in influenza type Aviruses. As examples, subtypes of influenza type A virus can be H1N1virus, H1N2 virus, H3N2 virus, H3N8 virus, H5N1 virus, H7N2 virus, H7N3virus, H7N7 virus and H9N2 virus. Further information on the nucleotidesequences of the genomes of flu viruses can be obtained from publiclyaccessible gene databases such as www.flugenome.org/. Furtherinformation on the nucleotide sequences of the genomes of influenza typeA viruses can also be obtained from publicly accessible gene databasessuch as GenBank, EMBL or LANL: e.g. J02144; J02146; J02148; J02151;V00603; V01099; V01104; V01106. Further information on the nucleotidesequences of the genomes of influenza type B viruses can also beobtained from publicly accessible gene databases such as GenBank, EMBLor LANL: e.g. J02094; J02095; J02096; K00423; K01395; M20168; M20170;M20172. Further information on the nucleotide sequences of the genomesof influenza type C viruses can also be obtained from publiclyaccessible gene databases such as GenBank, EMBL or LANL: e.g. K01689;M10087; M17700. Further information regarding attenuated flu virusvaccines can be found in HICKLING J. (A review of production forinfluenza virus vaccines, and their suitability for deployment indeveloping countries for influenza pandemic preparedness, Initiative forvaccine research, World Health Organisation (WHO) (2006);www.who.int/vaccine) and RUDENKO L. G. et al. (Vaccine 19(2-3), 308-318(2000)). Further information regarding attenuated cold-adapted andtemperature-sensitive flu virus vaccine such as for instance FluMist®(MedImmune Vaccines) can be found in GLEZEN W. (Expert Rev. Vaccines3(2):131-9 (2004)).

According to the invention, “Paramyxoviridae” include Avulaviruses,Henipaviruses, Morbilliviruses, Respiroviruses, Rubulaviruses,Pneumoviruses and Metapneumoviruses. Avulaviruses include Newcastledisease virus (NDV) also called avian paramyxovirus virus 1 (APMV-1).Henipaviruses include Hendra virus (HeV) and Nipah virus (NiV).Morbilliviruses include measles virus (MV) of strains: Edmonston Bstrain (ENDERS J. F. and T. C. PEEBLES, Proc. Soc. Exp. Biol. Med.86:277-286 (1954)), Edmonston A and B attenuated strains (GRIFFIN D. andBellini W., in B. Fields, D. Knipe et al. (ed.), Virology, vol. 2.,1267-1312, Lippincott-Raven Publishers, Philadelphia (1996)),Schwarz/Moraten attenuated strain (GRIFFIN D. and Bellini W., Measlesvirus, in B. Fields, D. Knipe, et al. (ed.), Virology, vol. 2.,1267-1312, Lippincott-Raven Publishers, Philadelphia (1996);commercialized by Aventis Pasteur under Rouvax™) and attenuated MVstrains as described in EP0540135, WO2008/086042 or WO99/49017.Respiroviruses include Sendai virus (SeV, also called murineparainfluenza virus 1), bovine parainfluenza virus 3 (BPIV-3), humanparainfluenza virus 1 (HPIV-1) and human parainfluenza virus 3 (HPIV-3).Rubulaviruses include human parainfluenza virus 2 (HPIV-2), humanparainfluenza virus 4a (HPIV-4a), human parainfluenza virus 4b (HPIV-4b)and mumps virus. Pneumoviruses include human respiratory syncytial virusA2 (HRSV-A2), human respiratory syncytial virus B1 (HRSV-B1) and humanrespiratory syncytial virus S2 (HRSV-S2). Metapneumoviruses includehuman metapneumovirus (HMPV).

As used throughout the entire application, “proteins” include:

-   -   antibodies (e.g. rituximab; trastuzumab; cetuximab; milatuzumab;        eculizumab; tocilizumab; nimotuzumab; golimumab; ramcirumab;        bapineuzumab; infliximab; bevacizumab; adalimumab; ranibizumab;        palivizumab; omalizumab; natalizumab; panitumumab; abciximab;        efalizumab; certolizumab; tocilizumab; ustenkinumab),    -   receptor ligands (e.g. hormones; cytokines; growth factors),    -   hormones (e.g. adrenocorticotropic hormone (ACTH); antidiuretic        hormone (ADH, vasopressin); atrial-natriuretic peptide (ANP);        calcitonin; cholecystokinin (CCK); corticotropin-releasing        hormone (CRH); glucagon; gonadotropin-releasing hormone (GnRH);        growth hormone releasing hormone (GHRH); oxytocin; secretin;        somatostatin; thyrotropin-releasing hormone (TRH);        erythropoietin (EPO); follicle-stimulating hormone (FSH); growth        hormone (GH); human chorionic gonadotropin (HCG); insulin;        Insulin-like growth factor-1 (IGF-1); luteinizing hormone (LH);        parathyroid hormone (PTH); prolactin (PRL); thrombopoitin;        thyroid-stimulating hormone (TSH); Testosterone;        dihydrotestosterone (DHT); andostenedione;        dehydroepiandrosterone (DHEA); testosterone; calciferol;        calcitriol; estradiol; cortisol; aldosterone; progesterone;        adrenaline (epinephrine); dopamine; melatonin; noradrenaline        (norepinephrine, NE); triiodothyronine (T3); thyroxine (T4);        serotonin),    -   cytokines (e.g. interleukins (IL-1; IL-2; IL-3; IL-4; IL-5;        IL-6; IL-7; IL-8; IL-9; IL10; IL-11; IL-12; IL13; IL-14; IL-15;        IL16; IL-17; IL-18; IL-19; IL-20; IL-21; IL-22; IL-23; IL-24;        IL-25; IL-26; IL-27; IL-28; IL-29; IL-30; IL-31; IL-32; IL-33);        chemokines (CC chemokine ligand 1 (CCL-1); CCL-2; CCL-3 (also        called macrophage inflammatory protein 1 alpha (MIP-1α)); CCL-4        (also called macrophage inflammatory protein 1 beta (MIP-1β));        CCL-5 (also called RANTES); CCL-6; CCL7; CCL-8; CCL-9; CCL-10;        CCL-11; CCL-12; CCL-13; CCL-14; CCL-15; CCL-16; CCL-17; CCL-18;        CCL-19; CCL-20; CCL21; CCL-22; CCL-23; CCL-24; CCL-25; CCL-26;        CCL-27; CCL-28; CXC chemokin 1 (CXCL-1); CXCL-2; CXCL-3; CXCL-4        (also called platelet factor 4 (PF4)); CXCL-5; CXCL-6; CXCL-7;        CXCL-8; CXCL-9; CXCL-10; CXCL-11; CXCL-12; CXCL-13; CXCL-14;        CXCL-15; CXCL-16; CXCL-17; C chemokin 1 (XCL-1); XCL-2; CX₃C        chemokin 1 (CX₃CL-1); interferons (IFN-alpha; IFN-beta;        IFN-gamma); tumor necrosis factors (TNF-alpha; TNF-beta)),    -   growth factors (e.g. epidermal growth factor (EGF); vascular        endothelial growth factor (VEGF); Bone morphogenetic proteins        (BMPs); Erythropoietin (EPO); Fibroblast growth factor (FGF);        granulocyte colony-stimulating factor (G-CSF);        granulocyte-macrophage colony-stimulating factor (GM-CSF); human        pluripotent granulocyte colony-stimulating factor (hPG-CSF);        macrophage colony-stimulating factor (M-CSF); growth        differentiation factor-9 (GDF9); macrophage migration inhibitory        factor (MIF); insulin-like growth factor (IGF); myostatin        (GDF-8); nerve growth factor (NGF); platelet-derived growth        factor (PDGF); thrombopoietin (TPO); transforming growth factor        alpha(TGF-α); transforming growth factor beta (TGF-β); placental        growth factor (PIGF)), antigens (e.g. antigens of the MHC;        leukocyte function associated antigen-1 (LFA-1)),    -   cell adhesion molecules (e.g. intercellular cell adhesion        molecule (ICAM-1); neural cell adhesion molecules (NCAMs);        vascular cell adhesion molecule (VCAM-1); platelet-endothelial        cell adhesion molecule (PECAM-1); nectins; synaptic cell        adhesion molecules (SynCAMs)),    -   blood clotting factors (e.g. Factor VIII; Factor IX; tPA),    -   enzymes (e.g. alpha-amylase; beta-amylase; cellulose;        beta-Glucanase; beta-glucosidase; dextranase; dextrinase;        dlucoamylase; hemmicellulase; pentosanase; xylanase; invertase;        lactase; naringinase; pectinase; pullulanase; acid proteinase;        alkaline protease; bromelain; pepsin; aminopeptidase;        endopeptidase; subtilisin; aminoacylase; glutaminase; lysozyme;        penicillin acylase; isomerase; alcohol dehydrogenase; catalase;        chloroperoxidase; peroxidase; acetolactate decarboxylase;        histidase; cyclodextrin glycosyltransferase),        fragments thereof and combinations thereof.

According to one special embodiment, the invention relates to the use ofthe immortalized avian cell line deposited at the European Collection ofCell Cultures (ECACC) under accession number 08060502 and derivativesthereof, for the production of viruses and proteins.

According to preferred embodiment, the invention relates to the use ofthe immortalized avian cell line deposited at the European Collection ofCell Cultures (ECACC) under accession number 08060502 and derivativesthereof, for the production of viruses and the said virus is aFlaviviridae, preferably selected in the group consisting of Yellowfever virus strain (YFV) and Japanese encephalitis virus strain (JEV),and more particularly in the group consisting of YFV 17D strain and JEVNakayama wild strain. Example 3 describes a preferred method forproducing YFV 17D strain and JEV Nakayama wild strain.

According to another preferred embodiment, the invention relates to theuse of the immortalized avian cell line deposited at the EuropeanCollection of Cell Cultures (ECACC) under accession number 08060502 andderivatives thereof, for the production of proteins, such as for examplethose selected from the group consisting of cytokines, antibodies andhormones, and even more particularly in the group consisting of IL-2,rituximab and erythropoietin (EPO). A preferred method for producingIL-2 is described in Example 5. A preferred method for producingrituximab is described in Example 6. A preferred method for producingEPO is described in Example 7.

According to another special embodiment, the invention relates to theuse of the immortalized avian cell line deposited at the EuropeanCollection of Cell Cultures (ECACC) under accession number 08060501 andderivatives thereof, for the production of viruses and proteins.

According to preferred embodiment, the invention relates to the use ofthe immortalized avian cell line deposited at the European Collection ofCell Cultures (ECACC) under accession number 08060501 and derivativesthereof, for the production of viruses and the said virus is aFlaviviridae, preferably selected in the group consisting of Yellowfever virus strain (YFV) and Japanese encephalitis virus strain (JEV),and more particularly in the group consisting of YFV 17D strain and JEVNakayama wild strain. Example 3 describes a preferred method forproducing YFV 17D strain and JEV Nakayama wild strain.

According to another preferred embodiment, the invention relates to theuse of the immortalized avian cell line deposited at the EuropeanCollection of Cell Cultures (ECACC) under accession number 08060501 andderivatives thereof, for the production of viruses and the said virus isa Poxviridae, preferably selected in the group consisting of Vacciniavirus strain Copenhagen (VV-COP) and Modified Vaccinia virus Ankara(MVA). Example 4 describes a preferred method for producing VV-COP andMVA.

According to another preferred embodiment, the invention relates to theuse of the immortalized avian cell line deposited at the EuropeanCollection of Cell Cultures (ECACC) under accession number 08060501 andderivatives thereof, for the production of proteins, such as for examplethose selected from the group consisting of cytokines, antibodies andhormones, and even more particularly in the group consisting of IL-2,rituximab and erythropoietin (EPO). A preferred method for producingIL-2 is described in Example 5. A preferred method for producingrituximab is described in Example 6. A preferred method for producingEPO is described in Example 7.

The present invention also relates to a method for producing a viruscomprising the steps of:

-   -   a) infecting an immortalized avian cell line deposited at the        European Collection of Cell Cultures (ECACC) under accession        number 08060502, with a virus; and    -   b) cultivating the infected avian cell line under conditions        which are enabling virus amplification.

The present invention further relates to a method for producing andpurifying a wild type, an attenuated and/or a recombinant Orthopoxvirus,comprising the following steps:

-   -   a) preparing a culture of packaging cells;    -   b) infecting the packaging cell culture with an Orthopoxvirus;    -   c) culturing the infected packaging cells until progeny        Orthopoxvirus is produced;    -   d) incubation in presence of one or more nucleases;    -   e) recovering the Orthopoxviruses from the culture supernatant        and/or the packaging cells;    -   f) adding monovalent salts to the Orthopoxviruses recovered in        step e) under suitable conditions to inhibit the nuclease(s)        activity and to avoid the adsorption of said Orthopoxviruses to        the anion exchange adsorbent in step g);    -   g) contacting the mixture obtained in step f) with an anion        exchange adsorbent under suitable conditions to allow the        capture of nucleic acids;    -   h) clarifying the mixture obtained in step g) under suitable        conditions to allow the withdrawal of the cellular debris;    -   i) washing of the anion exchange adsorbent with a solution        comprising monovalent salts under suitable conditions to recover        the remained Orthopoxviruses in the flow through;    -   j) concentrating the flow through obtained in step h) and the        flow through obtained in step i);    -   k) diafiltrating the fraction comprising the Orthopoxviruses        obtained in step j), wherein said packaging cells are Cairina        moschata immortalized avian cell lines comprising a nucleic acid        sequence coding a telomerase reverse transcriptase (TERT)        covered by patent application WO 2007/077256. Are particularly        preferred, the following immortalized avian cell lines:    -   T3-17490 as deposited at the European Collection of Cell        Cultures (ECACC) under accession number 08060502 (see FIGS. 2, 3        and 4) or a derivative thereof;    -   T6-17490 as deposited at the European Collection of Cell        Cultures (ECACC) under accession number 08060501 (see FIGS. 5, 6        and 7) or a derivative thereof.

According to preferred embodiment, the virus is a Flaviviridae,preferably selected in the group consisting of Yellow fever virus strain(YFV) and Japanese encephalitis virus strain (JEV), and moreparticularly in the group consisting of YFV 17D strain and JEV Nakayamawild strain. Example 3 describes a preferred method for producing YFV17D strain and JEV Nakayama wild strain.

According to another special embodiment, the invention relates to amethod for producing a virus comprising the steps of:

-   -   a) infecting an immortalized avian cell line deposited at the        European Collection of Cell Cultures (ECACC) under accession        number 08060501, with a virus; and    -   b) cultivating the infected avian cell line under conditions        which are enabling virus amplification.

According to preferred embodiment, the produced virus is a Flaviviridae,preferably selected in the group consisting of Yellow fever virus strain(YFV) and Japanese encephalitis virus strain (JEV), and moreparticularly in the group consisting of YFV 17D strain and JEV Nakayamawild strain. Example 3 describes a preferred method for producing YFV17D strain and JEV Nakayama wild strain.

According to another preferred embodiment, the produced virus is aPoxviridae, preferably selected in the group consisting of Vacciniavirus strain Copenhagen (VV-COP) and Modified Vaccinia virus Ankara(MVA). Example 4 describes a preferred method for producing VV-COP andMVA.

The avian cell lines of the invention are preferably infected at atemperature comprised between 30° C. and 37° C., and more preferably at37° C. as described in Example 3 and 4. Step a) of infection of the celllines of the invention with a virus is performed in an appropriate cellculture medium. The cell culture medium can be for instance Dulbecco'sModified Eagle's Medium (DMEM, Invitrogen) or Basal Medium Eagle (BME,Invitrogen) which can be optionally supplemented with e.g. serum (e.g.Fetal Calf Serum (FCS)) and/or amino acid(s) (e.g. L-Glutamine). Thecell culture medium can also be a medium free from animal product. Manymedia free from animal product have been already described and some ofthem are commercially available such as for instance 293 SFM II; 293-FCells, SFM Adapted; 293-H Cells, SFM Adapted; 293fectin™ TransfectionReagent; CD 293 AGT™; CD 293 Medium; FreeStyle™ 293 Expression System;FreeStyle™ 293 Medium; FreeStyle™ 293-F Cells, SFM Adapted; VP-SFM;VP-SFM AGT™; Adenovirus Expression Medium (AEM) Growth Medium forPER.C6® Cells; CD 293 AGT™; CD 293 Medium; COS-7L Cells, SFM Adapted;EPISERF® Medium; OptiPro™ SFM (all available from Invitrogen). Asdescribed in Example 3 and in Example 4, preferred cell culture mediumused for the infection of the avian cell lines of the invention is BME(Invitrogen) supplemented with FCS and L-Glutamine.

The cell lines of the present invention are infected with a virus at aMultiplicity of Infection (MOI) which depends on the produced virus. Forinstance, a Flaviviridae is seeded into the avian cell lines of theinvention at a MOI which is preferably comprised between about 0.001 and0.1. More particularly, when the produced Flaviviridae is YFV or JEV,the MOI is more preferably about 0.001 as described in Example 3, FIG.7A, FIG. 8A, FIG. 9A and FIG. 10A. As another example, a Poxviridae isseeded into the avian cell lines of the invention at a MOI which ispreferably comprised between about 0.0001 and 0.1. More particularly,when the produced Poxviridae is VV-COP, the MOI is more preferably about0.0001 as described in Example 4 and FIG. 11B. When the producedPoxviridae is a MVA, the MOI is more preferably about 0.05 as describedin Example 4 and FIG. 11A.

The infected cell lines are then cultivated under conditions which areenabling virus amplification (i.e. step b)) meaning that the viralgenome is transcribed, translated into viral proteins and packaged intoinfectious viral particles.

The infected cell lines can be cultivated as adherent cells to surfacesor in suspension, in presence or absence of (micro)carriers (aspreviously defined). Cell line culture can be done for instance indishes, roller bottles or in bioreactors, using batch, fed-batch,continuous systems, hollow fiber, and the like. The infected avian celllines of the invention are preferably cultivated at a temperaturecomprised between 30° C. and 37° C., and more preferably at 37° C. asdescribed in Example 3 and 4. Step b) of culture of the infected aviancell lines is performed in an appropriate cell culture medium which canbe the same or different from the cell culture medium used for theinfection of the avian cell lines (in step a)). The infected avian celllines are preferably cultivated for between 1 and 6 days depending onthe virus. For instance, when the produced virus is YFV, the infectedavian cell lines are more preferably cultivated for between 1 and 3 daysas shown in FIG. 7A and FIG. 9A. As another example, when the producedvirus is JEV, the infected avian cell lines are more preferablycultivated for between 1 and 4 days as shown in FIG. 8A and FIG. 10A. Asanother example, when the produced virus is VV-COP or a MVA, theinfected avian cell lines are more preferably cultivated between 1 and 4days as shown in FIG. 11(A-B).

According to the invention, step a) of infection can be preceded by astep of culturing a cell line of the invention in an appropriate cellculture medium which can be the same or different from the cell culturemedium used for the infection of the avian cell lines (in step a)) andfrom the cell culture medium used for the culture of the infected aviancell lines (in step b)). The avian cell lines are preferably cultivatedfor between 1 and 3 days, more preferably for 1 day before infection, ata temperature comprised between 30 and 37° C., more preferably at 37° C.as described in Example 3 and 4.

According to the invention, step b) of culture the infected cell linecan be followed by a step of incubation in presence of one or morenucleases (i.e. endonuclease or exonucleases) in order to degrade thenucleic acids (e.g. DNA; RNA) present in solution. Nucleases preferablyused according to the present invention are endonucleases. Endonucleasesthat can be used according to the invention can be classified based ontheir substrates as follows: deoxyribonucleases (DNases) which degradeDNA; ribonucleases (RNases) which degrade RNA; and endonucleases thatdegrade DNA and RNA. Endonucleases DNases include but are not limited toDNase I, DNase II and endodeoxyribonuclease IV. Endonucleases RNasesinclude but are not limited to RNase I, RNase III, RNAse E, RNAse F andRNAse P. Endonucleases that degrade DNA and RNA include but are notlimited to Benzonase®. Endonuclease preferably used according to thepresent invention is Benzonase®. Benzonase® degrades nucleic acid (e.g.DNA; RNA) by hydrolyzing internal phosphodiester bonds between specificnucleotides. Upon complete digestion, all free nucleic acids (e.g. DNA;RNA) present in solution are reduced to 5′-monophosphate terminatedoligonucleotides which are 3 to 8 bases in length. Benzonaze® has noproteolytic activity. Benzonaze® used according to the present inventionis preferably pharmaceutically acceptable. Pharmaceutically acceptableBenzonaze® are commercially available (e.g. Eurogentec under thereference ME-0280-10; Merck under the reference e.g. 1.01653.0001).

According to the invention, the concentration of nuclease(s) used is ina range of 5 to 100 U/ml, preferably in a range of 5 to 50 U/ml, andmore preferably 10 U/ml.

The viruses produced are then recovered from the supernatant and/or fromthe cells. When the produced viruses are recovered from the cells (i.e.from the cells only, or from the cells and from the supernatant), thestep of recovering of the viruses produced can be preceded by a stepallowing the disruption of the cell membrane. This step leads to theliberation of the viruses from the cells. The disruption of the cellmembrane can be induced by various techniques well known by the oneskilled in the art. These techniques comprise but are not limited tofreeze/thaw, hypotonic lysis, sonication (by using a sonicator) andmicrofluidization (by using a microfluidizer). Sonicators arecommercially available from e.g. Heraeus PSP, Biologics, Misonix orGlenMills. Preferred sonicators used according to the present inventionare SONITUBE 20 kHz type SM 20-120-3 and SONITUBE 35 kHz type SM35-400-3 (Heraeus PSP). Microfluidizers are commercially available frome.g. Microfluidics Corporation. The avian cell membrane can also bedisrupted by using a using a SLM Aminco French press. The cell membranecan also be disrupted by using a high speed homogenizer. High speedhomogenizers are commercially available from e.g. Silverson Machines orIke-Labotechnik.

According to special embodiment of the invention, the viruses recoveredare then purified. Purification of the viruses produced can comprise forinstance one or more of the following steps:

-   -   A clarification allowing under suitable conditions the        withdrawal of the cellular debris. Said clarification can be        performed by e.g. depth filtration. Depth filtration includes        but is not limited to the use of one or more commercially        available products such as Sartopure® filters from Sartorius        (e.g. Sartopure® PP2), CUNO Incorporated AP series depth filters        (e.g. AP01), CUNO Incorporated CP series depth filters (e.g.        CP10, CP30, CP50, CP60, CP70, CP90), CUNO Incorporated HP series        depth filters (e.g. HP10, HP30, HP50, HP60, HP70, HP90), CUNO        Incorporated Calif. series depth filters (e.g. CA10, CA30, CA50,        CA60, CA70, CA90), CUNO Incorporated SP series depth filters        (e.g. SP10, SP30, SP50, SP60, SP70, SP90), CUNO Delipid and        Delipid Plus filters, Millipore Corporation CE series depth        filters (e.g. CE15, CE20, CE25, CE30, CE35, CE40, CE45, CE50,        CE70, CE75), Millipore Corporation DE series depth filters (e.g.        DE25, DE30, DE35, DE40, DE45, DE50, DE55, DE560, DE65, DE70,        DE75), Millipore Corporation HC filters (e.g. A1HC, B1HC, COHC),        CUNO PolyNet™ Filters (e.g. PolyNet™ PB P050, P100, P200, P300,        P400, P500, P700), Millipore Clarigard and Polygard filters,        CUNO Life Assure filters, ManCel Associates depth filters (e.g.        PR 12 UP, PR12, PR 5 UP); and PALL or SeitzSchenk Incorporated        filters. In order to improve the clarification capacity of the        available depth filtration units, it can be useful to couple two        or more units with decreasing pore sizes. In this embodiment,        the mixture to be clarified passes through the first depth        filtration unit where the biggest contaminants are retained and        subsequently passes through the second depth filtration unit.        With this regard, the clarification is preferably performed by        depth filtration, and more preferably over filters having a pore        size 8 μm coupled to filters having a pore size of 5 μm.        Preferred filters having a pore size of 8 μm and 5 μm used        according to the present invention are Sartopure® filters        commercially available from Sartorius (Sartopure® PP2). The        depth filtration is preferably performed at a flow rate of 1        L/minute.    -   A concentration which can be performed by e.g. microfiltration        or ultrafiltration. Microfiltration is a pressure driven        membrane process that concentrates and purifies large molecules.        More specifically, a solution is passed through filters whose        pore size has been chosen to reject the virus in the retentate        and allow small molecules (e.g. proteins) to pass through the        filters into the permeate. Microfiltration reduces the volume of        the extraction solution. Filters used according to the invention        are preferably autoclavable commercially available filters such        as for instance Prostak Microfiltration Modules (Millipore).    -   A diafiltration which is an improvement of microfiltration (as        previously described) and involves diluting said fraction        comprising the virus with a solution to effect a reduction in        the concentration of the impurities in said fraction. The        dilution of the fraction comprising the viruses allows washing        out more of the impurities from said fraction. It is understood        that the diafiltration may be carried out in a batch mode,        semi-continuous mode, or a continuous mode. The diafiltration        can be advantageously used to change the buffer in which the        virus is comprised. For example, it can be useful to exchange        the buffer used in the purification process against a        pharmaceutically acceptable buffer. Filters used for the        diafiltration according to the invention allow the rejection of        the virus in the retentate and the passage of the small        molecules (e.g. proteins) through the filters into the permeate.        Such filters are preferably autoclavable commercially available        filters such as for instance Prostak Microfiltration Modules        (Millipore).    -   A chromatography using a cation or an anion exchange adsorbent,        and preferably an anion exchange adsorbent. The functional        groups of the anion exchange adsorbent can be primary,        secondary, tertiary or quaternary amino group such as for        instance dimethylaminoethyl (DMAE), diethylaminoethyl (DEAE),        trimethylaminoethyl (TMAE), triethylaminoethyl (TEAE), the group        —R—CH(OH)—CH₂—N+-(CH₃)₃ (also named Q group; see Streamline®        resins, Pharmacia) or other groups such as for instance        polyethyleneimine (PEI) that already have or will have a formal        positive charge within the pH range of 7.0 to 9.0. The anion        exchange adsorbent can consist in, but is not limited to, e.g. a        beads-formed matrix or a membrane. When the anion exchange        adsorbent consists in a beads-formed matrix, the matrix can be        e.g. agarose, hydrophilic polymer, cellulose, dextran or silica.        Chains (e.g. dextran chains) are coupled to the matrix.        Functional groups as previously described are attached to the        chains through chemically stable bonds (e.g. ether bonds). Anion        exchange adsorbents consisting of beads-formed matrix used        according to the invention are preferably autoclavable such as        for instance UNOsphere® Q (BioRad), UNOsphere® S (BioRad),        STREAMLINE™ Q Sepharose® XL (Amersham Biosciences), STREAMLINE™        SP Sepharose® XL (Amersham Biosciences) or BioSepra® Q hyperZ        (Pall Corporation).

When the anion exchange adsorbent consists in a membrane, the membraneused has a pore size lower than the size of the virus.

The wild type, attenuated, recombinant and/or temperature sensitivevirus produced can be further inactivated so that the outer virion coathas been left intact but the replicative function has been destroyed.Preparation of said “whole-killed virus” can take the route of heat orchemicals. The chemicals used include for formaldehyde orbeta-propiolactone and formalin (CHERTOVA E. et al., AIDS Vaccine(2001)).

According to one special embodiment, the invention relates to a methodfor producing proteins comprising the steps of:

-   -   a) contacting an immortalized avian cell line deposited at the        European Collection of Cell Cultures (ECACC) under accession        number 08060502, with at least one recombinant vector comprising        a nucleotide sequence coding at least one protein; and    -   b) cultivating the avian cell line under conditions which are        enabling the protein to be produced.

According to another special embodiment, the invention relates to amethod for producing a protein comprising the steps of:

-   -   a) contacting an immortalized avian cell line deposited at the        European Collection of Cell Cultures (ECACC) under accession        number 08060501, with a recombinant vector comprising a        nucleotide sequence coding the protein; and    -   b) cultivating the avian cell line under conditions which are        enabling the protein to be produced.

According to the present invention, the recombinant vector can be ofplasmid or viral origin and can, where appropriate, be combined with oneor more substances which improve the transfectional efficiency and/orstability of the vector. These substances are widely documented in theliterature which is available to the skilled person (see for exampleFEIGNER et al., Proc. West. Pharmacol. Soc. 32,115-121 (1987); HODGSONand SOLAIMAN, Nature Biotechnology 14, 339-342 (1996); REMY et al.,Bioconjugate Chemistry, 5, 647-654 (1994)). According to the presentinvention, the recombinant vectors are expression vectors. In a generalmanner, they are known to the skilled person and, while a number of themare available commercially, it is also possible to construct them or tomodify them using the techniques of genetic manipulation.

According to a preferred embodiment of the invention, the recombinantvector is a plasmid. Preferably, the plasmid which is used in thecontext of the present invention contains an origin of replication. Theplasmid can additionally comprise a selection gene which enables thetransfected cells to be selected or identified (complementation of anauxotrophic mutation, gene encoding resistance to an antibiotic, etc.).The plasmid can contain additional elements which improve itsmaintenance and/or its stability in the immortalized avian cell line ofthe invention. Examples of plasmids are described in Example 5, 6 and 7.

According to preferred embodiment of the invention, the produced proteinis a cytokine, more particularly an interleukin, and even moreparticularly IL-2. A preferred method for producing IL-2 is described inExample 5.

According to another preferred embodiment of the invention, the producedprotein is an antibody, and more particularly rituximab. Rituximab(Rituxan® or MabThera®) is a chimeric murine/human anti-CD20 monoclonalantibody. A preferred method for producing rituximab is described inExample 6.

According to another preferred embodiment of the invention, the producedprotein is a hormone, and more particularly erythropoietin (EPO). Apreferred method for producing EPO is described in Example 7.

According to a preferred embodiment of the invention, the methods forproducing proteins and viruses are free from animal products (except theavian cell lines of the invention) and suitable for an asepticindustrial-scale manufacturing process to ensure a full compliance withregulatory requirements regarding sterility of vaccines. As usedthroughout the entire application, <<animal product>> refer to anycompound or collection of compounds that was produced in or by an animalcell in a living organism.

The present invention also relates to a purified wild type, attenuated,recombinant and/or temperature sensitive virus obtained by the method aspreviously described.

The present invention also relates to a purified whole-killed virusobtained by the method as previously described.

The present invention also relates to a purified protein obtained by themethod as previously described.

The present invention also relates to a pharmaceutical composition, andmore particularly a vaccine, comprising a purified wild type,attenuated, recombinant, temperature sensitive and/or whole-killed virusobtained by the method as previously described.

The present invention also relates to a pharmaceutical compositioncomprising a purified protein obtained by the method as previouslydescribed.

As used herein, a “pharmaceutical composition” refers to a compositioncomprising a pharmaceutically acceptable carrier. Said pharmaceuticallyacceptable carrier is preferably isotonic, hypotonic or weaklyhypertonic and has a relatively low ionic strength, such as for examplea sucrose solution. Moreover, such a carrier may contain any solvent, oraqueous or partially aqueous liquid such as nonpyrogenic sterile water.The pH of the pharmaceutical composition is, in addition, adjusted andbuffered so as to meet the requirements of use in viva Thepharmaceutical compositions may also include a pharmaceuticallyacceptable diluent, adjuvant or excipient, as well as solubilizing,stabilizing and preserving agents. For injectable administration, aformulation in aqueous, nonaqueous or isotonic solution is preferred. Itmay be provided in a single dose or in a multidose in liquid or dry(powder, lyophilisate and the like) form which can be reconstituted atthe time of use with an appropriate diluent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts light microscopy imaging of the Cairina moschataimmortalized avian cell line ECACC 08060502 (passage 39).

FIG. 2 depicts the Cairina moschata immortalized avian cell line ECACC08060502 growth curve (from passage 7 to passage 75).

FIG. 3 depicts the Cairina moschata immortalized avian cell line ECACC08060502 population doubling time evolution (from passage 7 to passage75).

FIG. 4 depicts light microscopy imaging of the Cairina moschataimmortalized avian cell line ECACC 08060501 (passage 45).

FIG. 5 depicts the Cairina moschata immortalized avian cell line ECACC08060501 growth curve (from passage 15 to passage 51).

FIG. 6 depicts the Cairina moschata immortalized avian cell line ECACC08060501 population doubling time evolution (from passage 16 to passage51).

FIG. 7(A-B) depicts the Flaviviridae production profile (Yellow FeverVirus 17D(Mol 0.001)) of Cairina moschata immortalized avian cell lineECACC 08060502 (FIG. 7A) and of VERO cell line (FIG. 7B).

FIG. 8(A-B) depicts the Flaviviridae production profile (JapaneseEncephalitis Virus Nakayama wild strain (Mol 0.001)) of Cairina moschataimmortalized avian cell line ECACC 08060502 (FIG. 8A) and of VERO cellline (FIG. 8B).

FIG. 9(A-B) depicts the Flaviviridae production profile (Yellow FeverVirus 17D (Mol 0.001)) of the Cairina moschata immortalized avian cellline ECACC 08060501 (FIG. 9A) and of VERO cell line (FIG. 9B).

FIG. 10(A-B) depicts the Flaviviridae production profile (JapaneseEncephalitis Virus Nakayama wild strain(Mol 0.001)) of the Cairinamoschata immortalized avian cell line ECACC 08060501 (FIG. 10A) and ofthe VERO cell line (FIG. 10B).

FIG. 11(A-B) depicts the Poxviridae production profile (Vaccinia Virusstrain Copenhagen (VV-COP) (FIG. 11 B) and Modified Vaccinia virusAnkara (MVA) (FIG. 11A)) of the Cairina moschata immortalized avian cellline ECACC 08060501.

FIG. 12(A-C) depicts the production of rituximab from Cairina moschataimmortalized avian cell line ECACC 08060501 (FIG. 12C) and from CHO K1cell line (FIG. 12A) and from CHO DG44 cell line (FIG. 12B).

FIG. 13 depicts the production of erythropoietin (EPO) from Cairinamoschata immortalized avian cell line ECACC 08060502 (T3) and ECACC08060501 (T6).

To illustrate the invention, the following examples are provided. Theexamples are not intended to limit the scope of the invention in anyway.

EXAMPLES Example 1 Immortalized Cairina moschata cell line ECACC08060502

The Cairina moschata cells ECACC 08060502 (passage 39) have a homogenousfibroblast like morphology (FIG. 1). The static monolayer is stable upto 100% confluence and subject to contact inhibition. The cells weretested negative for mycoplasma contamination and for microbialcontamination as well. The cell line growth curve (from passage 7 topassage 75) (FIG. 2) shows a continuous exponential growth phase frompassage 19 to passage 75. Focusing on the evolution of the populationdoubling time (PDT), a progressive stabilisation and decrease isobserved, in particular it can be noted that the PDT is stabilised underthe 48 h mark during the 10 latest passages (FIG. 3). The correspondingnumber of population doublings (population doubling level, PDL) has beencalculated by cumulating the 2 exponential growth phases: during the 75passages the cells have undergone at least 147 population doublings(PD). The number of population doublings a primary cell can undergobefore entering senescence is tissue and specie dependent. It iscommonly admitted that the upper limit is situated between 50 and 60 PD.The Cairina moschata cell line ECACC 08060502 is therefore far beyondthe Hayflick limit and is consequently referred as immortalized cellline.

N.B.:

-   -   The population doubling level (PDL) refers to the number of cell        generations (biomass 2 fold increase). PDL calculation:        PDL=Ln(final/initial cell number)/Ln(2);    -   The population doubling time (PDT), also called generation time,        is the time needed for one population doubling. PDT calculation:        PDT=Δt*Ln(2)/Ln(final/initial cell number).

Example 2 Immortalized Cairina moschata cell line ECACC 08060501

The Cairina moschata cells ECACC 08060501 (passage 45) have a homogenousfibroblast like morphology (FIG. 4). The static monolayer is stable upto 100% confluence and subject to contact inhibition. The cells weretested negative for mycoplasma contamination and for microbialcontamination as well. The cell line growth curve (from passage 15 topassage 51) (FIG. 5) shows a continuous exponential growth phase frompassage 19. During this period the measured population doubling time(PDT) was progressively decreasing. Average PDT passed from 94 h(passage 20 to 35) to 52 h (passage 36 to 51) (FIG. 6). The number ofcalculated population doublings (PDL) corresponding to the 51 passagesis at least 71 population doublings.

The Cairina moschata cell line ECACC 08060501 is therefore far beyondthe Hayflick limit and is consequently referred as immortalized cellline.

N.B.

-   -   The population doubling level (PDL) refers to the number of cell        generations (biomass 2 fold increase). PDL calculation:        PDL=Ln(final/initial cell number)/Ln(2);    -   The population doubling time (PDT), also called generation time,        is the time needed for one population doubling. PDT calculation:        PDT=Δt*Ln(2)/Ln(final/initial cell number).

Example 3 Production of Flaviviridae

3.1 Production of Yellow Fever virus (YFV).

YFV amplification capacities of Cairina moschata immortalized avian celllines ECACC 08060502 and 08060501 were evaluated and compared to VEROcell line which is the reference for Flaviridae propagation. Cairinamoschata cell lines are grown in Basal Medium Eagle (Invitrogen)supplemented with 10% Foetal Calf Serum (FCS) and 4 mM L-Glutamine andthe VERO cell line in Dulbecco's Modified Eagle's Medium (Invitrogen)supplemented with 5% FCS. Infections were performed in the same culturemedia. The Yellow fever 17D vaccine strain (YFV 17D) (Stamaril™, SanofiPasteur) was evaluated in this experiment. Cairina moschata immortalizedavian cell lines ECACC 08060502 and 08060501 and VERO were seeded atrespectively 4.10⁵, 2.10⁵ and 6.10⁵ cells in 6 well plates andcultivated for 24 hours in humid atmosphere at 37° C., 5% CO₂. Themedium was then removed and cells infected at MOI 0.0001 with 250 μL YFV17D virus diluted in culture medium. After a 1 hour absorption step,remaining virus suspension was removed, cells washed thrice with PBS and2 mL medium were added to each well. Virus was recovered fromsupernatants after 24, 48 and 72 hours infection at 37° C., 5% CO₂, andclarified for titration. The infected cell layers were dissociated withtrypsin, washed and fixed on slides. Indirect immunofluorescenceanalysis was performed on the slides in order to determine percentage ofinfected cells.

Titrations were performed by immunohistochemical assay on VERO cellsinfected with logarithmic virus dilutions. Five days after infection,cells were fixed, incubated with specific polyclonal IA (Immune Ascite),and revealed with a secondary antibody labelled with peroxidase.Colorimetric reaction was obtained using DAB substrate. Stained foci ofinfection were observed and counted, and viral titers were calculated asfocus forming units (FFU) per ml.

Results: As depicted in FIG. 7(A-B), the viral titer obtained for YFV17D with Cairina moschata immortalized avian cell line ECACC 08060502(FIG. 7A) after only 24 hours is more than 3 log higher compared withthe viral titer obtained with VERO cell line (FIG. 7B). As depicted inFIG. 9(A-B), the viral titer obtained for YFV 17D with Cairina moschataimmortalized avian cell line ECACC 08060501 (FIG. 9A) after only 24 and48 hours is 4 log higher compared with the viral titer obtained withVERO cell line (FIG. 9B).

3.2 Production of Japanese Encephalitis virus (JEV).

JEV amplification capacities of Cairina moschata immortalized avian celllines ECACC 08060502 and 08060501 were evaluated and compared to VEROcell line which is the reference for Flavindae propagation. Cairinamoschata cell lines are grown in Basal Medium Eagle (Invitrogen)supplemented with 10% Foetal Calf Serum (FCS) and 4 mM L-Glutamine andthe VERO cell line in Dulbecco's Modified Eagle's Medium (Invitrogen)supplemented with 5% FCS. Infections were performed in the same culturemedia. The Japanese Encephalitis Nakayama wild strain (collection ofCentre National de la Recherche Scientifique (CNRS)) was evaluated inthis experiment. Cairina moschata immortalized avian cell lines ECACC08060502 and 08060501 and VERO were seeded at respectively 4.10⁵, 2.10⁵and 6.10⁵ cells in 6 well plates and cultivated for 24 hours in humidatmosphere at 37° C., 5% CO₂. The medium was then removed and cellsinfected at MOI 0.0001 with 250 μL JEV Nakayama wild strain diluted inculture medium. After a 1 hour absorption step, remaining virussuspension was removed, cells washed thrice with PBS and 2 mL mediumwere added to each well. Virus was recovered from supernatants after 24,48 and 72 hours infection at 37° C., 5% CO₂, and clarified fortitration. The infected cell layers were dissociated with trypsin,washed and fixed on slides. Indirect immunofluorescence analysis wasperformed on the slides in order to determine percentage of infectedcells.

Titrations were performed by immunohistochemical assay on VERO cellsinfected with logarithmic virus dilutions. Five days after infection,cells were fixed, incubated with specific polyclonal IA (Immune Ascite),and revealed with a secondary antibody labelled with peroxidase.Colorimetric reaction was obtained using DAB substrate. Stained foci ofinfection were observed and counted, and viral titers calculated asfocus forming units (FFU) per ml.

Results: As depicted in FIG. 8(A-B), the viral titer obtained for JEVNakayama wild strain with Cairina moschata immortalized avian cell lineECACC 08060502 (FIG. 8A) after 96 hours is more than 3 log highercompared with the viral titer obtained with VERO cell line (FIG. 8B). Asdepicted in FIG. 10(A-B), the viral titer obtained for JEV Nakayama wildstrain with Cairina moschata immortalized avian cell line ECACC 08060501(FIG. 10A) after 72 and 96 hours is respectively 1 log and 8 log highercompared with the viral titer obtained with VERO cell line (FIG. 10B).

Example 4 Production of Poxviridae

4.1 Production of Modified Vaccinia virus Ankara (MVA).

MVA amplification capacities of Cairina moschata immortalized avian celllines ECACC 08060502 and 08060501 were evaluated and compared to primarychicken embryo fibroblasts (CEFs) usually used as substrate for MVAproduction. An MVA (Collection Nationale de Cultures de Microorganismes(CNCM) under depositary N⁶⁰² I-721) expressing eGFP was chosen in orderto facilitate virus propagation follow up and titration. Cairinamoschata immortalized avian cell lines ECACC 08060502 and 08060501 andCEFs were seeded at 2.10⁶ cells in T-flask of 25 cm² and cultivated for24 hours in humid atmosphere at 37° C., 5% CO₂. The medium (Basal MediumEagle (BME) supplemented with 10% Foetal Calf Serum (FCS) and 4 mML-Glutamine) was then removed and cells infected at MOI 0.05 with 500 μLMVA virus diluted in PBS 1% cations, 1% FCS. After a 30 minutesadsorption step, remaining virus suspension was removed, cells washedonce with PBS and 5 mL BME 10% FCS were then added to each flask. Viruswas recovered by a freezing-thawing step from cells and supernatantafter 0, 24, 48, 72 and 96 hours infection at 37° C., 5% CO₂. Recoveredvirus suspensions were sonicated prior to titration in order to avoidaggregates.

Titrations were performed in triplicates on CEFs seeded in 6 cm culturedishes, infected with logarithmic virus dilutions and overlaid withagar. Plaque forming units of MVA, expressing eGFP, were visualized withfluorescent binocular and counted after 72 hours. The Cairina moschataimmortalized avian cell line ECACC 08060502 did not amplify MVA.

Results: As depicted in FIG. 11A, the Cairina moschata immortalizedavian cell line ECACC 08060501 enables MVA amplification comparable tothe one obtained with classical CEF substrate. The highest virus titerswere reached for both Cairina moschata immortalized avian cell lineECACC 08060501 and CEFs after 48 h infection. Concordantly, clearcytopathic effect was observed in both cells (data not shown).

4.2 Production of Vaccinia virus strain Copenhagen (VV-COP).

VV-COP amplification capacities of Cairina moschata immortalized aviancell lines ECACC 08060502 and 08060501 were evaluated and compared toprimary chicken embryo fibroblasts (CEFs) usually used as substrate forVV-COP production. A VV-COP strain (GOEBEL et al. 1990; Genbankaccession number M35027.1) comprising a defective J2R gene (see WEIR andMOSS 1983; Genbank accession number AAA48082) was used for thisexperiment. Cairina moschata immortalized avian cell lines ECACC08060502 and 08060501 and CEFs were seeded at 14.10⁶ cells in T-flask of175 cm² and cultivated for 24 hours in humid atmosphere at 37° C., 5%CO₂. The medium (Basal Medium Eagle (BME) supplemented with 10% FoetalCalf Serum (FCS) and 4 mM L-Glutamine) was then removed and cellsinfected at MOI 0.0001 with 5 mL VV-COP virus diluted in PBS 1% cations,1% FCS. After a 30 minutes adsorption step, 35 mL BME 10% FCS were addedto each flask. Virus was recovered by a freezing-thawing step from cellsand supernatant after 24, 48, 72 and 96 hours infection at 37° C., 5%CO₂. Recovered virus suspensions were sonicated prior to titration inorder to avoid aggregates.

Titrations were performed in triplicates on BHK21 cells seeded in 6 wellplates. Cells were infected with logarithmic virus dilutions and liquidculture media was added after 1 hour virus absorption. 24 hours later,media was removed and the cells overlaid with a mixture of crystalviolet and red neutral. After staining of the cells, plaque formingunits of virus were visualized with binocular and counted. The Cairinamoschata immortalized avian cell line ECACC 08060502 did not amplifyVV-COP.

Results: As depicted in FIG. 11B, the Cairina moschata immortalizedavian cell line ECACC 08060501 enables VV-COP amplification even if lessefficient compared to the amplification obtained with classical CEFsubstrate.

Example 5 Production of IL-2

Supernatants from Cairina moschata immortalized avian cell lines ECACC08060502 and 08060501 were transiently transfected with an IL-2 (CMVpromoter) expression plasmid (pTG8363: human IL2 cloned in pCiNeoplasmid, CMV promoter). Supernatants were collected after 24, 48, 72 and96 hours. IL-2 was quantified by ELISA (Quantikine, RD Systems) andfunctionality determined in a CTLL assay.

Results: Both cell lines ECACC 08060502 and 08060501 produced functionalIL-2. Amount was equivalent between both cell lines. Furthermore levelof production was independent from the transfection method used, rangingfrom 4865 IU/mL (CTLL) and 211 ng/mL (ELISA) corresponding to a specificactivity of 23.1 IU/ng, to 5672 IU/mL (CTLL) and 308 ng/mL correspondingto a specific activity of 18.4 IU/ng. So the measured specificactivities of IL-2 produced in the cells lines are at least equivalentto the 13.16 IU/ng WHO international standard for IL-2 (Human,Jurkat-derived).

Example 6 Production of Rituxan

Heavy and light chains were synthesized by GeneArt and correspondingexpression plasmid assembled based on the pCI-Neo vector (Promega).Transient gene expression was performed with specific <<low IgG FCS>>.The Cairina moschata immortalized avian cell lines ECACC 08060502 and08060501 were expanded 5 days in 10% low IgG FCS versus classic FCS. Nonegative effect on cell growth was observed. Supernatants from celllines ECACC 08060502 and 08060501 were collected and the amount ofmonoclonal antibody was evaluated using an IgG ELISA (total human IgG,Bethyl). Results: In transient and non-optimized conditions up to 0.5μg/per ml were produced in cell line ECACC 08060502 and up to 2.5 μg/perml in cell line ECACC 08060501. Rituxan transiently produced either inCHO standard cell lines (CHO K1 (FIG. 12A) and CHO DG44 (FIG. 12B) adihydrofolate reductase-deficient CHO clone) or in cell line ECACC08060501 (FIG. 12C) was purified in order to analyze and compare thecorresponding glycosylation patterns. Mass spectrometry analysis (FIG.12(A-C)) showed no fucosylation difference between CHO expressed Rituxanand cell line ECACC 08060501 expressed Rituxan.

Example 7 Production of Erythropoietin (EPO)

Expression transient assays were performed using a commerciallyavailable plasmid obtained from Invitrogen (GeneStorm® Human Clones, refHK1000 RG001720, Invitrogen) and in which the human EPO was undercontrol of a pCMV promoter.

Three transfection reagents were evaluated, in accordance with themanufacturer's instructions: Lipofectamine2000 (Invitrogen), Superfect(Qiagen) and Fugene6 (Roche); as well an electroporation device(Nucleofector, Basic Fibroblast kit, Amaxa). Supernatant from Cairinamoschata immortalized avian cell lines ECACC 08060502 and 08060501 werecollected after 48, 72 and 96 hours, and human EPO quantified by ELISA.

Results: In both cell lines, maximum expression level was obtained after96 hours using the Fugene6 transfection reagent followed by theNucleofector System (FIG. 13). In transient and non-optimized conditionsup to 0.5 μg/per ml were produced. All documents (e.g. patents, patentapplications, publications) cited in the above specification are hereinincorporated by reference. Various modifications and variations of thepresent invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments.

Indeed, various modifications of the described modes for carrying outthe invention which are obvious to those skilled in the art are intendedto be within the scope of the following claims.

1.-24. (canceled)
 25. Use of the immortalized avian cell line depositedat the European Collection of Cell Cultures (ECACC) under accessionnumber 08060502 and derivatives thereof, for the production of virusesand proteins.
 26. Use of the immortalized avian cell line deposited atthe European Collection of Cell Cultures (ECACC) under accession number08060501 and derivatives thereof, for the production of viruses andproteins.
 27. Use according to claim 25 or claim 26, wherein the proteinis selected in the group consisting of antibodies, receptor ligands,hormones, cytokines, growth factors, cell adhesion molecules, bloodclotting factors, enzymes, fragments thereof, and combinations thereof.28. Use according to claim 25 or claim 26, wherein the protein isselected in the group consisting of cytokines, antibodies, and hormones.29. Use according to claim 25 or claim 26, wherein the protein isselected in the group consisting of IL-2, rituximab, and erythropoietin(EPO).
 30. A method for producing a protein comprising the steps of: a)contacting an immortalized avian cell line deposited at the EuropeanCollection of Cell Cultures (ECACC) under accession number 08060502 witha recombinant vector comprising a nucleotide sequence coding theprotein; and/or b) cultivating the avian cell line under conditionswhich are enabling the protein to be produced.
 31. A method forproducing a protein comprising the steps of: a) contacting animmortalized avian cell line deposited at the European Collection ofCell Cultures (ECACC) under accession number 08060501, with arecombinant vector comprising a nucleotide sequence coding the protein;and/or b) cultivating the avian cell line under conditions which areenabling the protein to be produced. Page 5
 32. The method according toclaim 30 or claim 31, wherein the protein is selected in the groupconsisting of antibodies, receptor ligands, hormones, cytokines, growthfactors, cell adhesion molecules, blood clotting factors, enzymes,fragments thereof, and combinations thereof.
 33. The method according toclaim 30 or claim 31, wherein the protein is selected in the groupconsisting of cytokines, antibodies, and hormones.
 34. The methodaccording to claim 30 or claim 31, wherein the protein is selected inthe group consisting of IL-2, rituximab, and erythropoietin (EPO).